The proposed research involves study at the molecular level of nucleic acid-protein interactions through application of physical biochemical techniques to components of the E. coli lactose operon regulatory system. Fragments of DNA containing the lac control region have been prepared in quantity using recombinant DNA technology. The binding of RNA polymerase and of the catabolite activator protein (CAP) to these fragments will be characterized using optical methods and a sedimentation technique which permits evaluation of association constants. The same approach will be applied to the RNA polymerase-CAP-promoter system, with the aim of elucidating the molecular mechanism whereby RNA polymerase initiates, and CAP stimulates, transcription at catabolite sensitive operons. It is planned also to clone a mutant lac control region so that its interactions with proteins can be compared with those at the wild-type promoter. Studies will be continued of the binding of RNA polymerase and of CAP to "non-specific" DNAs (i.e., single- and double-stranded DNAs with various base compositions but lacking a specific control region). These experiments yield information about gross features of the DNA-protein interaction (e.g., is there preferential binding to the double helix, is there base sequence or base composition specificity, etc.?). Such studies using well-defined synthetic DNA sequences will be essential for interpreting data from experiments with the more complicated promoter sequence.